Method for Differentiating of Human Embryonic Stem Cells Into the Osteoblastic Lineage

ABSTRACT

Disclosed are a composition for introducing the osteogenic differentiation of human embryonic stem cells and a method for differentiating human embryonic stem cells into an osteoblastic lineage by inhibiting the mTOR signaling pathway. When cultured in the presence of an inhibitor of the mTOR signaling pathway, human embryonic stem cells are effectively induced to differentiate into an osteoblastic lineage. The osteogenic differentiation of human embryonic stem cells using the method and the composition is useful in examining the development and differentiation mechanism of osteoblasts and the cause of metabolic bone diseases, including osteoporosis. In addition, the method and the composition can be applied to the development of osteogenic differentiation techniques for generating clinically useful, terminally differentiated mature cells or progenitor cells.

TECHNICAL FIELD

The present invention relates to a method for differentiating humanembryonic stem cells into an osteoblastic lineage, using an inhibitoragainst the mTOR signaling pathway, and a composition for inducingosteogenic differentiation.

BACKGROUND ART

Stem cells, especially human embryonic stem cells, have recently arisenas a promising cell therapeutic in the regenerative medicine and medicalindustries. In recognition of the economic and industrial use and addedvalue of human embryonic stem cells, the development of technologies andmaterials for inducing human embryonic stem cells to differentiate intofunctionally specific cells has emerged as one of the most interestingtopics. As embryonic stem cells were first derived from mouse embryos in1981 by Evans and Kaufman, mouse embryonic stem cells have been used asbasic materials for use in the study of embryonic stem cells and thedevelopment of differentiation inducing technologies.

Embryonic stem cells possess two characteristic properties:self-renewal—the ability to go through numerous cycles of cell divisionunder ex vivo conditions without differentiation while maintaining anormal nucleus type; and potency—the capacity to differentiate, at leasttheoretically, into almost all specialized cell types that constitutethe body under culture conditions. These surprising properties greatlyrequire technologies and materials for the development and applicationof human embryonic stem cells. Since 1998, when a breakthrough in humanembryonic stem cell research came when Thomson first developed atechnique for isolating and growing cells when derived from the innercell mass of human blastocysts, active research has been conducted todevelop technologies targeting human embryonic stem cells, especiallytechnologies for tissue-specific differentiation-inducing technologies.Many research reports disclose successes in the differentiation of humanembryonic stem cells into retinal progenitor cells (Lamba D A et al.,2006), nerve cells (Li X J et al., 2006; Zhang S C et al., 2001),hematopoietic cells (Tian X et al., 2005; Kaufman D S et al., 2005;Kaufman D S et al., 2001), cardiomuscular cells (Kehat I et al., 2003;Kehat I et al., 2001), and pancreatic cells (Assady S et al., 2001)under ex vivo culture conditions, and the possibility of using humanembryonic stem cells as cell therapeutics was also suggested (Zhang S Cet al., 2001).

The bone maintains the homeostasis thereof through bone formation andremodeling. The site at which active bone remodeling takes place isknown as a bone remodeling unit (BRU) or bone multicellular unit, whichconsists of osteoblasts and osteoclasts, which play critical roles inosteogenesis and bone resorption, respectively. A hindrance tocooperation between the two cells in the remodeling process gives riseto various metabolic bone diseases, including osteoporosis. However,none of the therapies developed thus far ensure complete recovery frommetabolic bone diseases. Thus, there is still emphasis on theprophylaxis of metabolic bone diseases in the medical field.

Extensive efforts have recently been made to produce osteoblasts usingstem cell differentiation inducing techniques and to apply osteoblaststo the enhancement of bone tissue functions and the treatment of bonetissue injuries. As osteoblasts responsible for bone formation arenaturally derived from mesenchymal stem cells (Caplan A I, 1991),immense attention has been paid to the use of mesenchymal stem cells ininducing osteogenic differentiation and as cell therapeutics (Halleux Cat al., 2001; Jaiswal N et al., 1997; Hashimoto J et al., 2006; HofmannS et al., 2007; Xin X et al., 2007; Quarto R et al., 2001). Themesenchymal stem cells of adult tissues may be a useful approach to boneregeneration with autogenous bone grafts, but are disadvantageous inthat they are very small in number and difficult to collect by bonemarrow aspiration. Hence, the collected cells must be proliferated to anecessary population by ex vivo culturing. However, the fact that alimitation on the possible number of cell division cycles exists andthat there is a high possibility of inducing cell modification duringthe large number of cycles of cell division act as a bottleneck for theuse of mesenchymal stem cells. It was reported that nine or morepassages cause mesenchymal stem cells to experience aging and lose stemcell properties and osteogenetic potency (Bonab MM et al., 2006). On theother hand, early-stage human embryonic stem cells enjoy the advantageof being applicable to the treatment of various diseases not onlybecause they are a means of understanding the mechanism of osteogenicdifferentiation, but also because a relatively large number of the cellscan be supplied thanks to the ability thereof to proliferate throughnumerous cycles of cell division under ex vivo culture conditions.

Osteogenic supplements, such as ascorbic acid, β-glycerophosphate, anddexamethasone, are reported to be useful in the induction of osteogenicdifferentiation (Karp J M et al., 2006; Cao T et al., 2005; Bielby R Cet al., 2004; Sottile V et al., 2003). Also reported are inductionmethods for the differentiation of human embryonic stem cells intoosteoblasts by co-culturing with cells derived from bone tissues (Ahn SE et al., 2006), and the differentiation of human embryonic stemcell-derived mesenchymal stem cells into osteoblasts (Barberi T et al.,2005; US2005/0282274 A1). The transforming growth factor-beta (TGF-beta)subfamily, including activin, bone morphogenetic protein (BMP), inhibin,and growth/differentiation factor (GDF), is known to play a criticalrole in the formation and maintenance of bone tissues. Particularly,human embryonic stem cells are potentially induced to differentiate intoosteoblasts when cultured in the presence of BMP2or BMP4. Theunderstanding of the mechanism by which osteoblasts are differentiatedfrom embryonic stem cells and the techniques of inducing thedifferentiation have not advanced to a level sufficient to develop celltherapeutics having excellent clinical functions. In order to overcomethis, it is important to understand factors involved in osteoblastdifferentiation and their mechanisms. It is also important to findmaterials controlling the differentiation factors and utilize them inthe induction of osteoblast differentiation.

mTOR (mammalian target of rapamycin), a member of the PIKK(phosphoinositide kinase-related kinase) family, is an importantdownstream mediator in the PI3k/Akt signaling pathway, which is known toplay a critical role in controlling the proliferation anddifferentiation of embryonic stem cells. Rapamycin binds intracellularlyto FK506 binding protein-12 (FKBP12) and the rapamycin-FKBP12 complextargets mTOR, inhibiting its kinase activity, which in turn inhibits thephosphorylation and activation of the downstream translationalregulators p70S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E(eIF4E) binding protein 1 (4E-BP1). By phosphorylation, mTOR activatesthe downstream translational regulators, thus promoting variousintracellular functions including protein synthesis (Harris T E et al.,2003). Rapamycin, known as an immunosuppressive, is reported to have theactivity of inducing the differentiation of various cell lines includingmesenchymal stem cells into osteoblasts (Ogawa T et al., 1998; Tang L etal., 2002), and to have a therapeutic effect on osteolysis(US2006/0173033 A1). Also, recent findings suggest that phosphatidicacid, a competitor with rapamycin, activates mTOR, increasing theself-renewal of stem cells while 1-butanol and rapamycin, identified asantagonists of mTOR, inhibit mTOR activity, inducing the differentiationof stem cells (WO 2006/027545 A2, A3).

Many studies on human embryonic stem cells are conducted on the basis ofresults of research on mouse embryonic stem cells, but it is reportedthat there are differences between human and mouse embryonic stem cellsin proliferation and differentiation properties and relevant molecularregulation mechanisms. Intensive and thorough research into thedifferentiation of human embryonic stem cells, conducted by the presentinventors, resulted in the finding that human embryonic stem cells areinduced to effectively differentiate into an osteoblastic lineage in aculture medium supplemented with an inhibitor against mTOR, known toplay an important role in a cellular signaling pathway, leading to thepresent invention.

DISCLOSURE Technical Problem

It is therefore an object of the present invention to provide acomposition for inducing the differentiation of human embryonic stemcells into an osteoblastic lineage.

It is another object of the present invention to provide a method forinducing the differentiation of human embryonic stem cells into anosteoblastic lineage by culturing the cells in a culture mediumcomprising the composition.

Technical Solution

In order to accomplish the above objects, the present invention providesa composition for inducing the differentiation of human embryonic stemcells into an osteoblastic lineage, comprising an inhibitor of mTOR,which inhibits the signal transmission in which mTOR is involved, inaccordance with an aspect of the present invention. Preferably, theinhibitor of mTOR functions downstream or upstream of the mTOR signalingpathway, and is selected from among rapamycin, a PI3K inhibitor, an AKTinhibitor, and combinations thereof.

mTOR (mammalian target of Rapamycin), also named FKBP12rapamycin-associated protein (FRAP/RAFT/RAPT/SEP), is a serine/threonineprotein kinase, which is an evolutionarily conserved member ofphosphoinositol kinase-related kinase (PIKK). mTOR is involved in theregulation of cell growth through the initiation of gene translation inresponse to nutrients such as amino acids (mainly leucine), growthfactors, insulin and mitogens. mTOR initiates translation by activatingthe ribosomal p70S6k protein kinase (S6K1) and by inhibiting the eIF4 γinhibitor 4E-BP1. mTOR is thought to be involved in numerous additionalcellular functions including actin organization, membrane traffickingsecretion, protein degradation, protein kinase C signaling, ribosomebiogenesis and tRNA synthesis (Schmelzle T et al., 2000).

Preferably in the present invention, rapamycin, represented by thefollowing Chemical Formula 1, may be used as an inhibitor of mTORfunctioning downstream of the mTOR signaling pathway. Rapamycin, such asthat commercially available from Calbiochem, forms a complex with FK-506binding protein 12 (FKBP12). The interaction of this FKBP12-rapamycincomplex with mTOR inhibits the kinase activity of mTOR.

A PI3K inhibitor or an AKT inhibitor can be used as an inhibitor of mTORfunctioning upstream of the mTOR signaling pathway. LY294002 (a productfrom Calbiochem), represented by the following Chemical Formula 2, is aknown PIK3 inhibitor.

LY294002 inhibits PI3K, but does not inhibit PI4K, an EGR receptor, aPDGF receptor, an insulin receptor, an MAP kinase, an S6 kinase, etc.

An AKT inhibitor, as an inhibitor of mTOR functioning upstream of themTOR signaling pathway, may be exemplified by the compound representedby the following Chemical Formula 3, also commercially available fromCalbiochem. In the present invention, the expression “AKT inhibitor” isintended to refer to the compound of Chemical Formula 3.

As used herein, the term “human embryonic stem cell” is intended torefer to a cell culture derived in vitro from the inner cell mass ofhuman blastocysts of the fertilized eggs just before nidation, whichpossesses pluripotency, that is, ability to give rise to any mature celltype, and includes, in a broad sense, embryoid bodies derived therefrom.

The term “osteoblast”, as used herein, is intended to refer to amononucleate cell that is responsible for bone formation. Bone consistsof bone matrix and bone cells, which are further divided intoosteoblasts and osteoclasts. Bone is a dynamic tissue that is constantlybeing reshaped by osteoblasts, which build bone, and osteoclasts, whichresorb bone. Osteoclasts are a type of bone cell that removes bonetissue by removing the bone's mineralized matrix. Osteoblasts produce aprotein mixture known as osteoid, which is composed mainly of Type Icollagen. Osteoblasts are also responsible for mineralization of theosteoid matrix with calcium, magnesium, phosphorus, etc.

ADVANTAGEOUS EFFECTS

The composition for inducing stem cells to differentiate into anosteoblastic lineage and the method for differentiating stem cells intoan osteoblastic lineage using the composition are useful in thedevelopment of cell therapeutics for metabolic bone diseases. Also, thepresent invention contributes to molecular biological research into thesignaling pathway for the differentiation of human embryonic stem cellsinto an osteoblastic lineage, thus being useful in the discovery ofother novel bone-related factors. Providing terminally differentiatedmature cells, in addition, the present invention is applied to surgicaland pharmaceutical research into metabolic bone diseases. Thedifferentiated cells produced according to the method of the presentinvention find applications in various medical and pharmaceuticalfields, including biological assay systems for medical efficacy usingstem cells and assay systems for the medical efficacy of osteogenicinducers and bone tissue enhancements in stem cells.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic view illustrating the mechanism of action ofrapamycin, LY294002, and an AKT inhibitor in an intracellular signalpathway.

FIG. 2 illustrates a process of inducing human embryonic stem cells todifferentiate into osteoblasts in the presence of rapamycin alone or incombination with osteogenic supplements including β-glycerophosphate,dexamethasone and L-ascorbic acid in a schematic view (A), and shows theresulting differentiated cells in optical photographs (B).

FIG. 3 illustrates a process of inducing human embryonic stemcell-derived embryoid bodies to differentiate into osteoblasts bytreatment with rapamycin, alone or in combination with osteogenicsupplements including β-glycerophosphate, dexamethasone and L-ascorbicacid in a schematic view (A) and shows the resulting differentiatedcells in optical photographs (B).

FIG. 4 illustrates a process of dissociating human embryonic stemcell-derived embryoid bodies into single cells and inducing the singlecells to differentiate into osteoblasts in the presence of rapamycinalone or in combination with osteogenic supplements includingβ-glycerophosphate, dexamethasone and L-ascorbic acid in a schematicview (A), and shows the resulting differentiated cells in opticalphotographs (B).

FIG. 5 is a photograph showing the electrophoresis results of the RT-PCRproducts using the osteoblast-specific genes obtained from two differenthuman embryonic stem cell lines treated as in FIG. 2.

FIG. 6 is a set of photographs showing the electrophoresis results ofRT-PCR products using the osteoblast-specific genes obtained from humanembryonic stem cell-derived embryoid bodies treated with rapamycin (R)(A), the PI3K inhibitor LY294002 (LY) (B), and an AKT inhibitor (AKT),along with genes obtained from undifferentiated human embryonic stemcells (ES) and untreated human embryonic stem cell-derived embryoidbodies (C).

FIG. 7 is a chart including two sets of bar graphs showing the relativeexpression levels of osteoblast-specific genes obtained after the cellsof FIG. 3 had differentiated for one week (A) and two weeks (B), asdetermined by quantitative real-time PCR.

FIG. 8 is a chart including sets of bar graphs showing the relativeexpression levels of osteoblastic-specific genes obtained after twodifferent human embryonic stem cell lines were treated for three weeksas in FIG. 3, as determined by quantitative real-time PCR.

FIG. 9 is a chart of a set of immunofluorescent photographs showing thedifferentiated cells of FIG. 3, on which osteoblastic markers BSP and OCwere expressed.

FIG. 10 is a chart including a set of immunochemical photographs showingthe Alizarin Red S-positive, differentiated cells of FIG. 3.

FIG. 11 is a chart including a set of bar graphs showing the relativeexpression levels of osteoblastic-specific genes obtained after thehuman embryonic stem cells of FIG. 3 were treated with osteogenicsupplements including β-glycerophosphate, dexamethasone and L-ascorbicacid alone or in combination with rapamycin for two weeks, as determinedby quantitative real-time PCR.

FIG. 12 is a chart including a set of immunochemical photographs showingthe cells of FIG. 3 after they were differentiated in the presence ofosteogenic supplements alone or in combination with rapamycin andstained with Alizarin Red S.

BEST MODE

A better understanding of the present invention may be grasped withreference to the following examples, which are set forth to illustrate,but are not to be construed to limit the present invention.

Example 1 Culture of Human Embryonic Stem Cell

In order to keep them pluripotent, undifferentiated human embryonic stemcells were grown on a feeder layer of mitomycin C-treated mouseembryonic fibroblast (MEF) cells in a DMEM/F12 culture medium(Invitrogen, USA) supplemented with 20% knockout serum replacement(Invitrogen, USA), 0.1 mM non-essential amino acids (NEAA; Invitrogen,USA), 0.1 mM beta-mercaptoethanol, 4 ng/ml recombinant human fibroblastgrowth factor (FGF) basic (Invitrogen, USA), and 1×penicillin-streptomycin (Invitrogen, USA). One day before culturinghuman embryonic stem cells, MEF feeder cells maintained in DMEM(Invitrogen, USA) supplemented with 10% fetal bovine serum (Hyclone,USA), 0.1 mM NEAAs, 1× penicillin-streptomycin, and 0.5 mMbeta-mercaptoethanol were inactivated for 1.5 hrs with 10 μg/mlmitomycin C (Sigma, USA) and then plated at a density of 7.5×10⁴cells/cm² on tissue culture dishes coated with 0.1% gelatin (Sigma,USA). For subcultures, colonies of human embryonic stem cells weretriturated into smaller cell clumps using a tool made from a glassPasteur pipette and the cell clumps were placed at suitable spatialintervals on feeder cells. From two days after the cell clumps wereplaced on the feed layer, the culture medium was changed with a freshone with subculturing at intervals of 5 or 6 days.

Example 2 Preparation of Embryoid Bodies and Single Cells from HumanEmbryonic Stem Cells

In order to prepare consistent sizes of embryoid bodies, human embryonicstem cell colonies were sliced into cell clumps having dimensions ofabout 500×500 μm using a tool made from a glass pipette, and asuspension of the cell clumps was collected and transferred intobacterial culture dishes. The resulting embryoid bodies were dissociatedinto single cells by washing with phosphate buffered saline (PBS),adding a 1× trypsin-EDTA solution (TrypLE Express, Invitrogen),incubating at 37° C. for 1˜5 min, washing twice with PBS, and plating onMatrigel (BD biosciences, USA)-coated culture dishes.

Example 3 Differentiation of Human Embryonic Stem Cells and EmbryoidBodies into Osteoblasts

For osteogenic differentiation by adhesion culturing, the humanembryonic stem cells obtained in Example 1, after passage, were culturedin the culture medium of Example 1 for 2 days, and then in a culturemedium, free of recombinant human FGF basic, containing 0.1˜200 nMrapamycin, 0.01˜50 μM LY294002, 0.01˜20 μM AKT inhibitor, orconventional osteogenic supplements (10 mM β-glycerophosphate, 0.1 mMDexamethasone, 0.1 mM L-ascorbic acid) for 7 days, with dailyreplacement with a fresh embryonic stem cell culture medium (FIG. 2A).

For osteogenic differentiation by embryoid body formation, as shown inFIG. 3A, the human embryonic stem cells obtained in Example 1 were firstcultured in the stem cell culture medium of Example 1 for one day afterpassage, and the stem cell colony was sliced into cell clumps havingdimensions of 500×500 μm. After being collected as a suspension, thecell clumps were transferred into bacterial cell culture dishes andincubated for 5 days in a suspension culture manner. Afterwards, theembryoid bodies thus obtained were transferred to Matrigel (BDbiosciences, USA)-coated culture dishes, free of human recombinant FGFbasic, containing 0.1˜200 nM rapamycin, 0.01˜50 μM LY294002, 0.01˜20 μMAKT inhibitor, or osteogenic supplements (10 mM β-glycerophosphate, 0.1mM Dexamethasone, and 0.1 mM L-ascorbic acid) for three weeks in anadhesion culture manner to achieve osteogenic differentiation (FIG. 3A).

For osteogenic differentiation by single cell preparation, the humanembryonic stem cells obtained in Example 1 were first cultured in thestem cell culture medium of Example 1 for one day after passage, and thestem cell colony was sliced into cell clumps having dimensions of500×500 μm. After being collected as a suspension, the cell clumps weretransferred into bacterial cell culture dishes and incubated for 5 daysin a suspension culture manner. The embryoid bodies thus obtained werewashed with PBS, treated with 1× trypsin-EDTA (TrypLE Express,Invitrogen) at 37° C. for 1˜5 min, washed twice with PBS, and incubatedin a suspension culture manner in Matrigel-coated culture dishes, freeof recombinant human FGF basic, containing 0.1˜200 nM rapamycin, 0.01˜50μM LY294002 and 0.01˜20 μM AKT inhibitor, or osteogenic supplements (10mM β-glycerophosphate, 0.1 mM Dexamethasone, 0.1 mM L-ascorbic acid)(FIG. 4A).

Example 4 Detection of Osteoblastic Marker

<4-1> Reverse Transcriptase PCR (RT-PCR) and Quantitative Real-Time PCR

The differentiation of human embryonic stem cells into osteoblasts inExample 3 was examined by detecting the expression ofosteoblast-specific genes through RT-PCR and quantitative real-time PCR.For this, after treatment or non-treatment with rapamycin, LY294002, anAKT inhibitor or osteogenic supplements (10 mM β-glycerophosphate, 0.1mM Dexamethasone, and 0.1 mM L-ascorbic acid), cells were collectedaccording to differentiation stages and subjected to total RNA isolationusing a TRIzol reagent (Invitrogen, USA). The RNA was used as a templateto synthesize cDNA with a oligo(dT) primer in the presence of aSuperscript II reverse transcriptase, followed by PCR with variousprimers listed in Table 1, below.

TABLE 1 Accession Product Genes Nos. Sequences Size (bp) GAPDH NM_002046Forward GAAGGTGAAGGTCGGAGTC 226 Reverse GAAGATGGTGATGGGATTTC Oct4NM_002701 Forward GAAGGATGTGGTCCGAGTGT 243 Reverse GTGACAGAGACAGGGGGAAANanog NM_024865 Forward ACCAGAACTGTGTTCTCTTCCACC 334 ReverseGGTTGCTCCAGGTTGAATTGTTCC ALP NM_000478 Forward GGGGGTGGCCGGAAATACAT 543Reverse GGGGGCCAGACCAAAGATAG ALP* NM_000478 ForwardCCGTGGCAACTCTATCTTTGG 70 Reverse GATGGCAGTGAAGGGCTTCTT al type INM_000088 Forward ATGGATTCCAGTTCGAGTATGGC 246 collagen ReverseCATCGACAGTGACGCTGTAGG BMP2 NM_001200 Forward ACCCGCTGTCTTCTAGCGT 140Reverse CTCAGGACCTCGTCAGAGGG BMP4 NM_130851 ForwardTGTTCACCGTTTTCTCGACTC 243 Reverse TCAGGTATCAAACTAGCATGG BSP NM_004967Forward CAGTATGACTCATCCGAAG 280 Reverse CTCCTCTTCTTCTTCATCAC BSP*NM_004967 Forward AGAGGAAGCAATCACCAAAATGA 66 Reverse GCACAGGCCATTCCCAAACbfa1 NM_004348 Forward CGGCAAAATGAGCGACGTG 268 ReverseCACCGAGCACAGGAAGTTG Osteocalcin NM_199173 Forward CACTCCTCGCCCTATTGGC138 Reverse GCCTGGGTCTCTTCACTACCT Osteonectin NM_003118 ForwardAGCACCCCATTGACGGGTA 105 Reverse GGTCACAGGTCTCGAAAAAGC OsteopontinNM_000582 Forward ACTCGAACGACTCTGATGATGT 224 ReverseGTCAGGTCTGCGAAACTTCTTA Osteo- NM_002546 Forward AGCACCCTGTAGAAAACACAC195 protegerin Reverse ACACTAAGCCAGTTAGGCGTAA Osterix NM_152860 ForwardCCCAGGCAACACTCCTACTC 175 Reverse GGCTGGATTAAGGGGAGCAAA Osterix*NM_152860 Forward GCTCTGCTCCAAGCGCTTTA 55 Reverse GGTGCGCTGGTGTTTGCT

PCR results for the expression of osteoblast-specific genes of Example3, as seen in FIG. 5, indicate that the osteoblastic markers Cbfa-1,osteocalcin, and osteoprotegerin are expressed in each of twoindependently established cell lines cultured in the presence ofrapamycin in an adhesion culture manner. Also, osterix, BMP2, andosteonectin genes were observed to be expressed in both cell lines, butin different patterns. The genes were expressed at greater levels in thepresence of 20 nM than 10 nM of rapamycin. In addition, sensitivity tothe expression of osteoblast-specific genes, although varying with celllines, was observed to show the same overall direction. It isaccordingly understood that the efficient induction of human embryonicstem cells into osteogenic differentiation requires the addition ofrapamycin in an amount that is sufficient to induce differentiation butnot so high as to induce cytotoxicity.

With reference to FIG. 6, the expression of osteoblast-specific geneswas observed to differ in amount and type among the osteoblastsdifferentiated from embryoid bodies in the presence of rapamycin,LY294002 or an AKT inhibitor. When differentiated in the presence ofrapamycin, the osteoblasts expressed marker genes at the highestdiversity (BMP2, osteocalcin, Cbfa-2, osterix, osteoprotegerin, GATA2,CMP) and in amounts as great as or greater than when differentiated inthe presence of LY294002 or an AKT inhibitor. Of the osteoblast-specificgenes, osterix and osteoprotegerin were both expressed at high levels inthe osteoblasts differentiated in the presence of the three compounds.

As to the expression pattern of osteoblast-specific genes, it can beanalyzed by quantitative real-time PCR. As seen in FIG. 7, whendifferentiation was induced in the embryoid bodies for one week, theexpression of osteoblast-specific genes was increased 3.24-fold forCbfa1, 1.38-fold for BMP2, 6.54-fold for BSP, 1.84-fold for osteocalcin,and 2.89-fold for osteopontin in a rapamycin-specific manner. After twoweeks of differentiation culture, the expression level was increased2.11-fold for Cbfa-1, 1.59-fold for osterix, 1.34-fold for BMP2,1.18-fold for BMP4, 1.63-fold for BSP, 1.35-fold for α1 type I collagen,1.61-fold for osteocalcin, 2.18-fold for osteoprotegerin, 1.94-fold forosteonectin, and 5.81-fold for osteopontin in the presence of rapamycin(FIG. 7). Also, it was observed that osterix, BMP2, BMP4, α1 type Icollagen, osteocalcin, osteoprotegerin, osteonectin, and osteopontincontinued to increase in expression level even after three weeks ofdifferentiation culture (FIG. 8). In response to rapamycin, both of thehuman embryonic stem cell lines were similarly increased in theexpression levels of osterix, BMP4, α1 type I collagen, osteoprotegerin,and osteopontin, but decreased in the expression level of Cbfa1 afterthree weeks of differentiation culture. As for BMP2, BSP, osteocalcinand osteonectin, however, their expression levels were measured todiffer from one cell line to the other. Referring to FIG. 11, theexpression of osteoblast-specific genes in the cells differentiated inthe presence of conventional osteogenic supplements (10 mMβ-glycerophosphate, 0.1 mM Dexamethasone, 0.1 mM L-ascorbic acid) aloneor in combination with rapamycin was quantitatively analyzed. After oneweek of differentiation culture with the osteogenic supplements, theexpression level was increased 4.54-fold for Cbfa1, 2.22-fold forosterix, 2.21-fold for BMP2, 1.93-fold for BMP4, 2.62-fold for BSP, and2.91-fold for osteoprotegerin. On the other hand, differentiationinduction with the osteogenic supplements in combination with rapamycinresulted in an increase in the expression level not only of Cbfa1 by2.27 times, osterix by 2.22 times, BMP2 by 2.21 times, BMP4 by 1.93times, BSP by 2.62 times, and osteoprotegerin by 2.91 times, but also ofα1 type I collagen by 1.82 times, osteocalcin by 3.36 times, andosteoprotegerinby 2.91 times, neither of which showed response to theosteogenic supplements.

<4-2> Analysis for Mineralization by Alizarin-Red S Staining

The cells obtained using the three differentiation inducing techniquesdescribed in Example 3 were analyzed for mineralization using anAlizarin-Red S staining method. After aspirating the culture medium, thecells on the culture dish were washed with PBS and fixed at 4° C. for 1hr with 70% ethanol. Thereafter, the cells were stained with a 40 mMalizarin red (AR) solution (pH 4.2) for 10 min, washed with distilledwater, and observed for mineral nodule formation under an opticalmicroscope before they were completely dried. As seen in FIG. 10, thecells which were induced to differentiate by rapamycin were positivelystained with Alizarin-Red. When cultured in the co-presence ofconventional osteogenic supplements (10 mM β-glycerophosphate, 0.1 mMDexamethasone, 0.1 mM L-ascorbic acid) and rapamycin, a relativelyhigher number of the cells were stained with Alizarin Red (FIG. 12). Aculture period of three weeks was required to maximize the number ofAlizarin Red-positive cells upon osteogenic differentiation withrapamycin alone. The co-presence of osteogenic supplements (10 mM3-glycerophosphate, 0.1 mM Dexamethasone, 0.1 mM L-ascorbic acid) andrapamycin allowed the cells to be visualized with Alizarin Red S in anearlier time.

<4-3> Immunofluorescent Staining

The osteogenic differentiation of the cells obtained using the threedifferentiation inducing techniques described in Example was identifiedby immunofluorescent staining. First, the cells were placed on a 1 cm²cover glass and the medium was aspirated. Following washing with PBS,the cells were incubated with a fixing solution(citrate-acetone-formaldehyde) for about 30 sec at room temperature andwashed with distilled water for 45 sec. Next, treatment with 3% bovineserum albumin/phosphate buffered saline (BSA/PBS) for 30 min wasfollowed by incubation with a primary antibody for 12 hrs. Then, thecells were washed three times for 15 min in 0.1% Triton X-100/PBS beforelabeling by incubation with an Alexa488-conjugated secondary antibody(Molecular probes, USA) for 30 min at room temperature. The cells werewashed again three times in 0.1% Triton X-100/PBS for 15 min each timefor observation under a fluorescent microscope (Olympus, Japan). Inorder to identify osteogenic differentiation, anti-bone sialoprotein(anti-BSP, Chemicon; 1:100), and anti-osteocalcin (R&D Systems; 1:500)were used for immunofluorescence. As seen in FIG. 9, visualization withan antibody against the late osteoblastic marker bone sialoprotein (BSP)or osteocalcin showed that when induced by rapamycin, the embryonic stemcells were differentiated into osteoblasts in the same or greater numberthan when induced by the conventional osteogenic supplements.Accordingly, it is understood that rapamycin is as good as or betterthan the conventional osteogenic supplements in terms of its ability toinduce osteogenic differentiation.

MODE FOR INVENTION

In an embodiment of the present invention, as shown in FIG. 1, humanembryonic stem cells (FIG. 2) or embryoid bodies (FIGS. 3 and 4) derivedfrom human embryonic stem cells were induced to differentiate into anosteoblastic lineage in the presence of rapamycin, LY294002 or an AKTinhibitor, which is known to inhibit a downstream signaling molecule inthe PI3K signaling pathway and thus play a critical role in controllingthe proliferation of human embryonic stem cells. The osteogenicdifferentiation, as seen in FIGS. 5 to 8, was identified asquantitatively analyzed for the expression of osteoblast-specific genesby RT-PCR. Osteoblastic markers were found to increase in expressionlevel when human embryonic stem cells were cultured in culture mediacontaining 10 mM LY294002, 0.1 mM AKT inhibitor, or 20 nM rapamycin for5 days, as illustrated in Stage II of FIG. 2, or when embryoid bodiesderived from human embryonic stem cells were cultured in culture mediacontaining 10 mM LY294002, 0.1 mM AKT inhibitor, or 20 nM rapamycin forthree weeks, as illustrated in Stage II of FIG. 3, indicating that thesethree compounds have the ability to induce osteogenic differentiation.Of the compounds, rapamycin was observed to have the most potentinduction activity, even at low doses (FIG. 6). After embryoid bodiesderived from human embryonic stem cells were cultured for three weeks inthe presence of 20 nM rapamycin, as illustrated in Stage II of FIG. 3,the cells were positively stained with both Alizarin Red S, which isspecific for calcium phosphate-deposited osteoblasts (FIG. 10), andimmunofluorescent dye with antibodies to the later osteoblastic makersBSP and osteocalcin, indicating that rapamycin is able to induceembryoid bodies to differentiate into mature osteoblasts.

In a concrete embodiment of the present invention, osteogenicdifferentiation was assayed when human embryonic stem cells werecultured: 1. in the presence of rapamycin, known as an mTOR inhibitor,only; 2. in the presence of conventional osteogenic supplements(β-glycerophosphate, Dexamethasone, and L-ascorbic acid), known toeffectively induce osteogenic differentiation; and 3. in the co-presenceof rapamycin and osteogenic supplements (β-glycerophosphate,Dexamethasone, L-ascorbic acid). Although it is a single chemical,rapamycin showed osteogenic differentiation activity as good as orbetter than that of the conventional osteogenic supplements in terms ofcell morphology (FIG. 2) and the expression level of osteoblasticmarkers (FIGS. 7 and 8). The most efficient differentiation into matureosteoblasts was detected when embryoid bodies derived from humanembryonic stem cells were treated with 20 nM rapamycin for three weeks,as illustrated in Stage II of FIG. 3. When using osteogenic supplements(β-glycerophosphate, Dexamethasone, and L-ascorbic acid) and rapamycinin combination, a culture period of two weeks was sufficient to expressosteoblastic markers (FIG. 11) and induce differentiation into matureosteoblasts (FIG. 12).

Meanwhile, as seen in FIG. 3, embryoid bodies were prepared from humanembryonic stem cells and induced to differentiate into osteoblasts. Inthis regard, first, human embryonic stem cells were grown to colonies ina fresh culture medium which was replaced 48 hrs after passage, and thecolonies were sliced into cell clumps having dimensions of about 500×500μm. These cell clumps were cultured in a suspension culture manner toform embryoid bodies which were then incubated with LY294002 (0-50 μM),an AKT inhibitor (0-20 μM), or rapamycin (0-200 nM) for 1˜3 weeks.Compared to a control, which was not treated with the inhibitors, theembryoid bodies treated with the inhibitors were found to increase inthe expression level of osteoblast-specific genes (FIGS. 5 to 8). Inboth cases, in which embryoid bodies derived from human embryonic stemcells were attached to culture dishes, as schematically illustrated inFIG. 3A, and in which single cells dissociated from the embryoid bodieswere attached to culture dishes, as schematically illustrated in FIG.4A, it was observed that osteogenic differentiation took placeefficiently. The single cell dissociation is expectedly useful in thequantitative and qualitative analysis for properties of differentiatedcells.

In accordance with an aspect of the present invention, a composition forinducing the osteogenic differentiation of human embryonic stem cellsmay be preferably provided as a medium composition. The mediumcomposition may comprise an inhibitor of mTOR, that is, rapamycin,LY294002, and/or an AKT inhibitor in addition to basic additives orconventional osteogenic supplements. The basic additives include sera,amino acids, antibiotics, differentiation-inhibiting factors, etc.Rapamycin is known as an immunosuppressive, like FK506 and cyclosporine.Hence, FK506 and cyclosporine are also contained in the composition forinducing the osteogenic differentiation of human embryonic stem cells.

The medium composition in accordance with the present inventionpreferably contains an mTOR inhibitor, for example, LY294002, at aconcentration from 0.1 to 50 μM, an AKT inhibitor at a concentrationfrom 0.1 to 20 μM, or rapamycin at a concentration from 0.1 to 200 nM.More preferably, the concentration of the mTOR inhibitor ranges from 0.1to 20 μM for LY294002, from 0.1 to 10 μM for an AKT inhibitor or from0.1 to 100 nM for rapamycin.

In accordance with another aspect thereof, the present inventionprovides a method for the osteogenic differentiation of human embryonicstem cells using the composition containing the mTOR inhibitor.

In the present invention, the osteogenic differentiation can be achievedusing an adhesion culture method, an embryoid body formation method, ora single cell dissociation method. The adhesion culture method isdirected to the differentiation of human embryonic stem cells while theyare attached to culture dishes. In the embryoid body formation method,embryoid bodies are derived from stem cells one day after passage, andare induced to differentiate into osteoblasts. As for the single celldissociation method, embryoid bodies are also formed as in the embryoidbody formation method and on day 4 after the formation, the embryoidbodies are dissociated into single cells which are then cultured in theadhesion culture manner.

First, a description is given of the adhesion culture method. As seen inFIG. 2A, human embryonic stem cells are co-cultured with mitomycinC-treated MEF (mouse embryonic fibroblast) feeder cells in a nutrientDMEM/F12 medium (Invitrogen, USA) containing 20% knockout serumreplacement (Invitrogen, USA), 0.1 mM non-essential amino acids (NEAA;Invitrogen, USA), 0.1 mM beta-mercaptoethanol, 4 ng/ml recombinant humanFGF basic (Invitrogen, USA), and 1× penicillin-streptomycin (Invitrogen,USA) for two days (Stage I). Afterwards, the human embryonic stem cellsare induced to differentiate into osteoblasts by being cultured for fivedays in a medium that is the same except that it is free of recombinanthuman FGF basic and is supplemented with LY294002, an AKT inhibitor,rapamycin, and/or conventional osteogenic supplements (10 mM3-glycerophosphate, 0.1 mM Dexamethasone, and 0.1 mM L-ascorbic acid)(Stage II).

Osteogenic differentiation using the embryoid body formation method isschematically illustrated in FIG. 3A. Initially, human embryonic stemcells are cultured for one day in the same manner as in the adhesionculture method, after which the colonies thus grown are sliced into cellclumps using a tool made from a glass pipette. The cell clumps arecultured for four days in a culture medium (80% knockout DMEM, 20% fetalbovine serum, 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, 1 mMnon-essential amino acids) to form embryoid bodies (Stage I), followedby culturing the embryoid bodies for an additional three weeks in thepresence of LY294002, an AKT inhibitor, rapamycin, and/or osteogenicsupplements (10 mM β-glycerophosphate, 0.1 mM Dexamethasone, and 0.1 mML-ascorbic acid) to induce osteogenic differentiation (Stage II).

Turning to FIG. 3A, osteogenic differentiation by single celldissociation is schematically illustrated. As in the adhesion culturemethod and the embryoid body formation method, embryoid bodies areprepared one day after the passage of human embryonic stem cells and arecultured for four days in the embryoid body culture medium (Stage I). Onday 4 after culturing, the embryoid bodies are dissociated into singlecells using a trypsin-EDTA solution (TrypLE Express, Invitrogen),followed by adhesion culturing for an additional three weeks in the samemedium in the presence of LY294002, an AKT inhibitor, rapamycin, and/orosteogenic supplements (10 mM β-glycerophosphate, 0.1 mM Dexamethasone,0.1 mM L-ascorbic acid) (Stage II).

The osteogenic differentiation of human embryonic stem cells by thethree differentiation methods can be monitored using reversetranscriptase PCR (RT PCR), quantitative real-time PCR,immunofluorescent staining, and Alizarin Red S staining.

As seen in the photograph of FIG. 5, taken of the gel on which RT-PCRproducts were electrophoresed, when differentiated with rapamycin, twoindependently established cell lines were found to express distinctivelyhigher levels of the osteoblastic markers Cbfa-1, Osteocalcin andOsteoprotegerin at higher levels, compared to an undifferentiatedcontrol. The expression levels of Osteocalcin and Osteonectin werehigher at a dose of 20 nM than 10 nM. Hence, when 20 nM of rapamycin wasadded to a culture medium, the osteoblast-specific genes were mosteffectively expressed, with no cytotoxic effects occurring.

Referring to FIG. 6, the cells differentiated by the embryoid bodyformation method illustrated in FIG. 3A were assayed for osteogenicdifferentiation through RT-PCR for osteoblast-specific genes. As seen inFIG. 6, LY294002, an AKT inhibitor, and rapamycin were all observed toincrease the expression levels of osteoblast-specific genes. Above andbeyond the LY294002 and AKT inhibitor, rapamycin was found to moreeffectively induce embryoid bodies to differentiate into osteoblasts, asqualitatively and quantitatively analyzed using osteoblastic markers(BMP2, Osteocalcin, Cbfa-2, Sterix, Osteoprotegerin, GATA2, CMP). Theseresults therefore indicate that the down-regulation of the mTORsignaling pathway (that is, inhibiting mediators downstream of mTOR)rather than the up-regulation (that is, inhibiting mediators upstream ofmTOR, such as PI3K or AKT) is more effective in the induction ofosteogenic differentiation.

The ability of rapamycin to induce osteogenic differentiation was alsoconfirmed using molecular biological methods, such as quantitativereal-time PCR)(FIG. 7) and immunofluorescent staining (FIG. 10), andAlizarin Red S staining, specific for osteoblasts. After one week ofincubation of embryoid bodies with rapamycin, as illustrated in FIG. 3A,osteoblastic markers were observed to increase in expression level (FIG.7), but it took an additional three weeks of incubation with rapamycinto obtain a mature osteoblast phenotype.

Finally, the cells differentiated using a single cell dissociationmethod, in which embryoid bodies are formed from human embryonic stemcells and dissociated into single cells, are assayed for osteogenicdifferentiation as measured by quantitative real-time PCR(FIG. 8) andimmunofluorescent staining (FIG. 11).

In order to induce osteogenic differentiation, rapamycin, LY294002, andan AKT inhibitor were used at various concentrations in culture media.It was observed that osteoblast-specific genes were efficientlyexpressed in the presence of 10 μM of LY29400, 1 μM of an AKT inhibitor,or 220 nM of rapamycin without the occurrence of cytotoxicity. The cellsderived from human embryonic stem cells in this way are osteoblastprogenitor cells which can be grown to produce osteoblasts in largequantities.

When cultured in the medium composition comprising an mTOR inhibitor,for example, LY294002, at a concentration ranging from 0.1 to 50 μM, anAKT inhibitor at a concentration from 0.1 to 20 μM, or rapamycin at aconcentration from 0.1 to 200 nM, human embryonic stem cells can beinduced to differentiate into osteoblasts. More preferably, theconcentration of the mTOR inhibitor ranges from 0.1 to 20 μM forLY294002, from 0.1 to 10 μM for an AKT inhibitor, or from 0.1 to 100 nMfor rapamycin.

INDUSTRIAL APPLICABILITY

Osteoblasts differentiated from human embryonic stem cells in accordancewith the method of the present invention can be used for cell therapyfor bone-related diseases.

1. A method for differentiating human embryonic stem cell or embryoidbodies derived from human embryonic stem cells, comprising culturing thehuman embryonic stem cells in a culture medium containing an mTORinhibitor or an inhibitor against an mTOR signaling pathway.
 2. Themethod according to claim 1, wherein the mTOR inhibitor or the inhibitoragainst the mTOR signaling pathway is selected from a group consistingof rapamycin, represented by the following Chemical Formula 1, a PI3Kinhibitor, and an AKT inhibitor:


3. The method according to claim 2, wherein the PI3K inhibitor is acompound represented by the following Chemical Formula 2:


4. The method according to claim 2, wherein the AKT inhibitor is acompound represented by the following Chemical Formula 3:


5. The method according to claim 2, wherein the rapamycin is containedat a concentration of 0.1˜100 nM in the culture medium.
 6. The methodaccording to claim 3, wherein the compound of Chemical Formula 2 iscontained at a concentration of 0.1˜20 μM in the culture medium.
 7. Themethod according to claim 4, wherein the compound of Chemical Formula 3is contained at a concentration of 0.1˜10 μM in the culture medium. 8.The method according to one of claims 1 to 7, wherein the culture mediumfurther contains an osteogenic supplement selected from a groupconsisting of β-glycerophosphate, dexamethasone, L-ascorbic acid, andcombinations thereof.
 9. The method according to one of claims 1 to 7,wherein the embryoid bodies derived from human embryonic stem cellsinclude embryonic stem cell clumps having a size of 500×500 μm.
 10. Themethod according to one of claims 1 to 7, wherein the embryoid bodiesderived from human embryonic stem cells are dissociated into singlecells.
 11. A composition for inducing human embryonic stem cells orembryoid bodies derived from human embryonic stem cells to differentiateinto osteoblasts, comprising at least one of the compounds representedby the following Chemical Formulas 1 to 3:


12. A culture medium composition for inducing human embryonic stem cellsor embryoid bodies derived from human embryonic stem cells todifferentiate into osteoblasts, comprising a compound of ChemicalFormula 1 at a concentration of 0.1˜100 nM, a compound of ChemicalFormula 2 at a concentration of 0.1˜20 μM, a compound of ChemicalFormula 3 at a concentration of 0.1˜10 μM, or a combination thereof(wherein Chemical Formulas 1 to 3 are as defined in claim 11).
 13. Theculture medium composition according to claim 12, comprising thecompound of Chemical Formula 1 at a concentration of 0.1˜20 nM (whereinChemical Formula 1 is as defined in claim 11).
 14. The culture mediumcomposition according to claim 12, comprising the compound of ChemicalFormula 2 at a concentration of 0.1˜10 μM (wherein Chemical Formula 2 isas defined in claim 11).
 15. The composition according to claim 12,comprising the compound of Chemical Formula 3 at a concentration of0.1˜5 μM (wherein Chemical Formula 3 is as defined in claim 11).
 16. Thecomposition according to one of claims 12 to 15, further comprising 10mM β-glycerophosphate, 0.1 mM dexamethasone, and 0.1 mM L-ascorbic acid.